Vaccine composition and use of surfactants as adjuvants of immunity

ABSTRACT

A composition comprising an aqueous solution comprising:
         (i) at least one antigen or at least one in vivo generator of a compound comprising an amino acid sequence, and   (ii) as an adjuvant of immunity, a surfactant, or a mixture of surfactants, having an overall HLB number of between 5 and 15.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional application of U.S. applicationSer. No. 09/698,121, filed Oct. 30, 2000, now U.S. Pat. No. 7,422,748,which claims the benefit under 35 U.S.C. §119 of FR 9913618 filed inFrance on Oct. 29, 1999, the disclosures of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

(i) Field of the Invention

The present invention relates to novel adjuvants for vaccinecompositions, and to compositions comprising at least one antigen, inparticular an antigen of viral, bacterial or parasitic origin, and atleast one adjuvant.

(ii) Description of the Related Art

The development of inactivated vaccines or vaccines containing purifiedantigens is increasingly significant, since it makes it possible toavoid adverse side effects. However, the improvement in the quality ofthe antigens occurs to the detriment of their immunogenic nature. It isfor this reason that they are combined with adjuvants of immunity.

Adjuvants of immunity are products which increase the reactions of theimmune system, when they are administered in the presence of antigens ofviral, bacterial or synthetic origin. They cause a massive appearance ofmacrophages at the site of injection, and then in the lymph nodes,increase the production of specific immunoglobulins, antibodies, andstimulate many cells involved in immune defense mechanism.

These adjuvants are diverse in nature. They can, for example, consist ofliposomes or emulsions.

Very effective Freund's adjuvants: they result from the combination of amineral oil and of a mannitol ester, possibly containing a killedmycobacterium. Vaccines prepared by mixing in equal parts a Freund'sadjuvant with an aqueous antigenic medium are still used as standardsthroughout the world for laboratory studies. They are in the form ofwater in oil (W/O) emulsions, i.e., emulsions in which the continuousphase is the oil. These emulsions are very viscous; they are thusdifficult to inject; they are also relatively unstable, since phasedisplacements are observed only a few days after their preparation.

By way of ordinary adjuvants, there are also metal salts, such asaluminum hydroxide, cerium nitrate, zinc sulphate, colloidal ironhydroxide or calcium chloride. Of these, aluminum hydroxide is the mostcommonly used. These adjuvants are described in the article by Rajesh K.Gupta et al., “Adjuvants, balance between toxicity and adjuvanticity”Vaccine, Vol. 11, Issue 3, 1993, pages 993-1006. They exhibit, however,weak immunostimulatory effectiveness, and sometimes induce, when thesetherapeutic compositions are injected, the formation of lesions andother local reactions, such as granulomas, at the point of injection.

More recently, it has been discovered that water-soluble salts ofdivalent or trivalent metals are good adjuvants of immunity, inparticular manganese gluconate, calcium gluconate, manganeseglycerophosphate, soluble aluminum acetate and aluminum salicylate. Suchadjuvants are described in the international patent applicationspublished under the numbers WO 96/32964 and WO 98/17311.

As other adjuvants of immunity, in particular in the case of mucosaladministration, mention may be made of the sympathomimetic compoundsdescribed in the international patent application published under thenumber WO 98/15288.

SUMMARY AND OBJECTS OF THE INVENTION

In the course of research into the development of novel adjuvants, theapplicant has discovered that some surfactants themselves exhibitimmunostimulatory effectiveness, and that it is thus possible to prepareaqueous vaccine compositions essentially free of oily phase, comprisingone or more of these agents as an immunostimulant.

It is for this reason that the subject of the present invention is acomposition in the form of an aqueous solution comprising:

-   -   (i) at least one antigen or at least one in vivo generator of a        compound comprising an amino acid sequence, and    -   (ii) as an adjuvant of immunity, a surfactant, or a mixture of        surfactants, having an overall HLB number of between 5 and 15.

The term “antigen” or the phrase “at least one in vivo generator of acompound comprising an amino acid sequence” refers to either killedmicroorganisms, such as viruses, bacteria or parasites, or purifiedfractions of these microorganisms, or living microorganisms whosepathogenic power has been attenuated. As a virus which can constitute anantigen according to the present invention, mention may be made ofrabies virus, herpes viruses, such as the virus of Aujeszky's disease,orthomixoviruses such as Influenzae, picornaviruses such as the virus offoot-and-mouth disease, or retroviruses such as HIVs. As microorganismsof the bacterial type which can constitute an antigen according to thepresent invention, mention may be made of E. coli, and those of thePasteurella, Furonculosis, Vibriosis, Staphylococcus and Streptococcusgenera. As parasites, mention may be made of the Trypanosoma, Plasmodiumand Leishmania genera. Mention may also be made of recombinant viruses,in particular nonenveloped viruses such as adenoviruses, the vacciniavirus, the canarypox virus, herpes viruses or baculoviruses. Referenceis also made to a living nonenveloped viral recombinant vector, thegenome of which contains, inserted preferably into a portion which isnonessential for the replication of the corresponding enveloped virus, asequence encoding an antigenic subunit which induces synthesis ofantibodies and/or a protective effect against the above-mentionedpathogenic enveloped virus or microorganism; these antigenic subunitscan be, for example, a protein, a glycoprotein, a peptide or a fractionwhich is a peptide and/or which is protective against an infection witha living microorganism such as an enveloped virus, a bacterium or aparasite. The exogenous gene inserted into the microorganism can be, forexample, derived from an HIV or Aujeszky virus.

Mention may be made in particular of a recombinant plasmid consisting ofa nucleotide sequence, into which is inserted an exogenous nucleotidesequence originating from a pathogenic microorganism or virus. The aimof the latter nucleotide sequence is to allow the expression of acompound comprising an amino acid sequence, the aim of this compounditself being to trigger an immune reaction in a host organism.

The expression “in vivo generator of a compound comprising an amino acidsequence” refers to an entire biological product capable of expressingsaid compound in the host organism into which said in vivo generator hasbeen introduced. The compound comprising the amino acid sequence can bea protein, a peptide or a glycoprotein. These in vivo generators aregenerally obtained by methods derived from genetic engineering. Moreparticularly, they can consist of living microorganisms, generally avirus, playing the role of recombinant vector, into which is inserted anucleotide sequence, in particular an exogenous gene. These compoundsare known in themselves, and are used in particular as recombinantsubunit vaccines. In this respect, reference may be made to the articleby M. Eloit et al., Journal of Virology (1990) 71, 2925-2431, and to theinternational patent applications published under the numbersWO-A-91/00107 and WO-A-94/16681. The in vivo generators according to theinvention can also consist of a recombinant plasmid which comprises anexogenous nucleotide sequence, and which is capable of expressing, in ahost organism, a compound comprising an amino acid sequence. Suchrecombinant plasmids and their method of administration to a hostorganism were described in 1990 by Lin et al., Circulation 82: 2217,2221; Cox et al., J. of Virol., September 1993, 67, 9, 5665-5667, and inthe international application published under the number WO 95/25542.Depending on the nature of the nucleotide sequence included in the invivo generator, the compound comprising the amino acid sequence which isexpressed within the host organism can:

(i) be an antigen, and enable the triggering of an immune reaction; (ii)have a curative action with respect to a disease, essentially a diseaseof a functional nature, which has been triggered in the host organism.In this case, the in vivo generator enables gene therapy type treatmentof the host.

By way of example, such a curative action can consist of synthesis bythe in vivo generator of cytokines, such as interleukins, in particularinterleukin-2. These interleukins allow the triggering or thereinforcement of an immune reaction directed towards selectiveelimination of cancerous cells.

A composition according to the invention comprises an antigenconcentration which depends on the nature of this antigen and on thenature of the individual treated. It is, however, particularlynoteworthy that an adjuvant according to the invention makes it possibleto notably decrease the conventional antigen dose required. The suitableantigen concentration can be determined conventionally by personsskilled in the art, Generally, this dose is about 0.1 μg/cm³ to 1 g/cm³,more generally between 1 μg/cm³ and 100 mg/cm³.

The concentration of said in vivo generator in the composition accordingto the invention depends, here again, in particular on the nature ofsaid generator and of the host in which is administered. Thisconcentration can be easily determined by persons skilled in the art, onthe basis of routine experiment. By way of indication, it may, however,be specified that, when the in vivo generator is a recombinantmicroorganism, its concentration in the composition according to theinvention can be between 10² and 10¹⁵ microorganisms/cm³, preferablybetween 10⁵ and 10¹² microorganisms/cm³. When the in vivo generator is arecombinant plasmid, its concentration in the composition according tothe invention can be between 0.01 and 100 g/dm³.

For the purpose of the present invention, the HLB number is calculatedusing the formula HLB=20 (1−I_(s)/I_(a)) in which I_(s) represents thesaponification index and I_(a) represents the acid index of saidsurfactant or of said mixture of surfactants. These two indices,saponification and acid indices, are determined by methods described inthe European Pharmacopoeia.

The main subject of the invention is a composition as defined above, inwhich the surfactant(s) is (are) chosen from modified fatty substancesand, preferably, the surfactants(s) is (are) chosen from modified fattysubstances having an overall HLB number of between 6 and 14.

The modified fatty substances used in the context of the presentinvention can be of mineral, plant or animal origin. As modified fattysubstances of mineral origin there are oils of petroleum origin. Asmodified fatty substances of plant origin, there are modified plantoils, for example modified groundnut, olive, sesame, soya bean,wheatgerm, grapeseed, sunflower, castor, flax, corn, copra, palm,walnut, hazelnut or rapeseed oils. As modified fatty substances ofanimal origin, there are, for example, modified squalane, modifiedsqualene, modified spermaceti oil or modified tallow oil.

The term “modified fatty substances” refers in particular to thealkoxylated derivatives of fatty substances, and more particularly thealkoxylated derivatives of oils or the alkoxylated derivatives of alkylesters of oils, and more particularly the ethoxylated and/orpropoxylated derivatives of oils or the ethoxylated and/or propoxylatedderivatives of the methyl, ethyl, linear or branched propyl, or linearor branched butyl esters of said oils. A subject of the invention ismore specifically a composition as defined above, in which the modifiedfatty substance is chosen from the ethoxylated derivatives of oilshaving a number of EOs of between 1 and 60.

A subject of the invention is particularly a composition as definedabove, in which the modified fatty substance is an alkoxylatedderivative of corn oil, or a mixture of alkoxylated derivatives of cornoil, having an overall HLB number of between 10 and 14, or a compositionas defined above in which the modified fatty substance is an ethoxylatedderivative of castor oil, or a mixture of alkoxylated derivatives ofcastor oil, having an overall HLB number of between 7 and 10. Asexamples of such compositions, there is the composition in which themodified fatty substance is chosen from the ethoxylated derivatives ofcorn oil having a number of EOs of between 20 and 40, or the compositionin which the modified fatty substance is a mixture of ethoxylatedderivatives of castor oil having a number of EOs equal to 7 and ofethoxylated derivatives of castor oil having a number of EOs equal to60.

A composition which is a subject of the present invention containsbetween 0.2 mg/cm³ and 500 mg/cm³ of adjuvant, more particularly between2 mg/cm³ and 500 mg/cm³ of adjuvant and preferably between 50 mg/cm³ and200 mg/cm³ of adjuvant.

According to a second specific aspect of the present invention, asubject of this invention is a composition as defined above, in whichthe surfactant(s) is (are) chosen from the alkoxylated derivatives ofesters of fatty acids and of polyols or the alkoxylated derivatives ofethers of fatty alcohols and of polyols, and more particularly fromalkoxylated fatty acid triglycerides, the polyglycerol alkoxylatedesters of fatty acids, the alkoxylated esters of fatty acids with ahexol, such as for example sorbitol or mannitol, or the alkoxylatedesters of fatty acids with a hexol anhydride, such as sorbitan ormannitan.

As fatty acids which are suitable for preparing these modified esters,there are more particularly those comprising from 12 to 22 carbon atoms,advantageously a fatty acid which is liquid at 20° C., such as forexample those comprising from 16 to 18 carbon atoms, for instance oleicacid, ricinoleic acid or isostearic acid.

The composition as defined above contains in particular one or moreethoxylated derivatives of esters of fatty acids and of polyols, or theethoxylated derivatives of ethers of fatty alcohols and of polyols,having a number of EOs of between 1 and 60. The surfactant, or themixture of surfactants, of this composition as defined above has moreparticularly an overall HLB number of between 10 and 14, and preferablybetween 12 and 13. As an example of such a composition, there is the onein which the surfactant is an ethoxylated derivative of mannitan oleatehaving a number of Eos of between 5 and 15, and preferably between 7 and11.

A surfactant according to the invention is preferably pharmaceuticallyacceptable for the mucous membranes; it must, in particular, be devoidof heavy metals and have very low acid or peroxide indices. It is alsodesirable for it to satisfy the standards of innocuity tests such asthose described by S. S. Berllin, Annals of Allergy, 1962, 20, 473, orthe abnormal toxicity tests described in the European Pharmacopoeia.

The composition according to the invention can comprise a conventionalimmunostimulant such as AVRIDINE®,N,N-dioctadecyl-N′,N′-bis(2-hydroxy-ethyl)propanediamine, MDP (muramyldepeptide) derivatives, in particular threonyl-MDP, mycolic acidderivatives or Lipid A derivatives.

The composition according to the invention can comprises one or morewater-soluble metal cation organic salts, such as for example calciumgluconate, manganese gluconate, aluminum salicylate or soluble aluminumacetate. When the adjuvant composition according to the inventioncomprises a pharmaceutically acceptable salt, this salt is at aconcentration of 0.02 to 3000 mg/cm³, preferably 0.1 to 1000 mg/cm³,more preferably from 0.1 to 150 mg/cm³.

The composition according to the invention can comprise asympathomimetic compound. The term “sympathomimetic compounds” refers inparticular to amphetamines, catecholamines, phenylisopropylamines andtyramine. As examples of such compounds mention may be made inparticular of isoproterenol, L-adrenalin, levarterenol, ephedrine,phenylephedrine and salbutamol When the adjuvant composition accordingto the invention comprises a sympathomimetic compound, this compound isat a concentration of 10⁻¹⁰ molar to 10⁻² molar, preferably from 10⁻⁷molar to 10⁻⁵ molar.

The use of surfactants as defined above as adjuvants in the vaccinecompositions, and more particularly in the vaccine compositions whichhave no oily phase, constitutes another aspect of the present invention.

The composition according to the invention can be used as a preventiveor curative medicinal product. Depending on the nature of the antigen orof the in vivo generator, a composition according to the invention canbe administered to fish, crustaceans such as shrimps, poultry, inparticular geese, turkeys, pigeons and chickens, to Canidae such asdogs, to Felidae such as cats, to pigs, to primates, to Bovidae, toOvidae and to horses. The composition according to the invention canalso be administered to humans. The administration of the compositioncan be carried out conventionally via the parenteral route, inparticular by subcutaneous, intramuscular or intraperitoneal injection,or via the mucosal route, in particular orally, rectally, nasally orvaginally. According to another aspect of the invention, it consists ofthe use of an adjuvant as defined above for preparing a vaccine intendedfor preventing or for treating an infectious disease, in particular aninfectious disease engendered by a virus or a microorganism, such asthose mentioned above.

According to another final aspect of the present invention, it consistsof the use of this adjuvant for preparing a composition intended totreat a disease of a functional nature, such as cancer or cysticfibrosis.

EXAMPLE 1

100 microliters of various compositions containing a surfactant,phosphate buffer (PBS) and 10 mg/cm³ of ovalbumin were injectedsubcutaneously into various batches of 5 female mice of the OF1 strain,weighing an average of 18 to 20 grams, at t=0 with a booster at t=28days.

Blood samples are taken at 14, 28, 42, 56, 90 and 180 days.

ELISA assays are carded out on the blood samples, for IgG1s in order todetermine the humoral immune response, and IgG2as in order to determinethe cellular immune response. Local reactions were evaluated at 7 daysand at 35 days.

The compositions are as follows:

Surfactant used (SA) Buffer Antigen (Composition) HLB of SA in (PBS) (10mg/cm³) in weight % SA μl in μl μl Ethoxylated corn oil 4.1 100 1900 20(3 EOs) (Reference 1) Ethoxylated corn oil 7.9 100 1900 20 (10 EOs) +glycerol at 2% of initial load (Composition A) Ethoxylated corn oil 10.4100 1900 20 (20 EOs) + glycerol at 2% of initial load (Composition B)Ethoxylated corn oil 12.3 100 1900 20 (30 EOs) + glycerol at 2% ofinitial load (Composition C) Ethoxylated corn oil 13.8 100 1900 20 (40EOs) + glycerol at 2% of initial load (Composition D) Ethoxylated cornoil 14.2 100 1900 20 (20 EOs) + glycerol at 4% of initial load(Composition E) Ethoxylated corn oil 11.3 100 1900 20 (40 EOs) +glycerol at 4% of initial load (Composition F) Mannitan oleate (5 EOs)10.9 100 1900 20 (Composition G) Mannitan oleate (8 EOs) 12.4 100 190020 (Composition H) Mannitan oleate (10 EOs) 13.1 100 1900 20(Composition I) Mannitan oleate (15 EOs) 14.6 100 1900 20 (CompositionJ) Mannitan oleate (20 EOs) 15.6 100 1900 20 (Composition K) Mannitanoleate (40 EOs) 17.3 100 1900 20 (Composition L) Mannitan oleate 3.3 1001900 20 (Reference 2) Mannitan oleate (8 EOs) 12.1 100 1900 20(Composition M) Mannitan oleate + 6.5 100 1900 20 mannitan oleate (8EOs) (Composition N) Mannitan oleate + 5.0 100 1900 20 mannitan oleate(8 EOs) (Composition O) Manganese gluconate — 200 1800 20 (Reference 3)Control — 0 2000 20

The results of the ELISA assays are as follows:

IgG1 assay (timescale in days) Composition D14 D28 D42 D56 D90 D180Reference (1) 1500 1000 32000 48000 32000 6000 Composition (A) 1000 10008000 12000 3000 1500 Composition (B) 1000 1000 64000 64000 16000 8000Composition (C) 2000 1000 96000 128000 128000 12000 Composition (D) 15001000 6000 32000 64000 6000 Composition (E) 1000 1000 32000 64000 9600024000 Composition (F) 3000 8000 64000 128000 128000 32000 Composition(G) 2000 2000 8000 64000 48000 8000 Composition (H) 4000 8000 128000128000 48000 16000 Composition (I) 4000 1000 128000 96000 48000 12000Composition (J) 1000 2000 64000 24000 6000 2000 Composition (K) 10001000 24000 12000 2000 2000 Composition (L) 1000 1000 18000 6000 20002000 Reference (2) 1000 1000 32000 16000 3000 2000 Composition (M) 40004000 128000 128000 256000 48000 Composition (N) 1500 1000 128000 6400032000 12000 Composition (O) 1000 1000 32000 20000 16000 2000 Reference(3) 32000 32000 256000 128000 32000 8000 Control 1000 1000 4000 20003000 1000

IgG2a assay (timescale in days) Composition D14 D28 D42 D56 D90 D180Reference (1) 1000 1000 1000 1500 3000 1000 Composition (A) 1000 10001000 1000 2000 1000 Composition (B) 1000 1000 2000 2000 2000 1000Composition (C) 1000 1000 4000 1500 3000 1000 Composition (D) 1000 10001000 2000 4000 1000 Composition (E) 1000 1000 1000 2000 4000 1000Composition (F) 1000 1000 1000 6000 4000 1000 Composition (G) 1000 10001000 8000 3000 100 Composition (H) 1000 1000 1000 3000 3000 1000Composition (I) 1000 1000 4000 3000 2000 1000 Composition (J) 1000 10003000 1500 2000 1000 Composition (K) 1000 1000 1000 1500 2000 1000Composition (L) 1000 1000 1000 1500 2000 1000 Reference (2) 1000 10001000 1500 3000 1000 Composition (M) 1000 1000 4000 1500 6000 1000Composition (N) 1000 1000 4000 1500 2000 1000 Composition (O) 1000 10004000 1500 6000 1000 Reference (3) 1000 1000 4000 16000 2000 1000 Control1000 1000 1000 1000 1000 1000

EXAMPLE 2

The procedure is carried out in the same way as in Example 1, with thefollowing ethoxylated castor oils as surfactants:

Surfactant used (SA) Buffer Antigen (Composition) HLB of SA (PBS) (10mg/cm³) weight % SA in μl in μl in μl Ethoxylated castor oil 6 100 190020 100% (7 EOs) (Composition P) Ethoxylated castor oil 7 100 1900 2089.13% (7 EOs) + 10.87% (60EOs) (Composition Q) Ethoxylated castor oil 8100 1900 20 78.26% (7 EOs) + 21.74% (60EOs) (Composition R) Ethoxylatedcastor oil 9 100 1900 20 67.39% (7 EOs) + 32.61% (60EOs) (Composition S)Ethoxylated castor oil 10 100 1900 20 56.52% (7 EOs) + 43.487% (60 EOs)(Composition T) Ethoxylated castor oil 10.6 100 1900 20 50% (7 EOs) +50% (60 EOs) (Composition U) Ethoxylated castor oil 11 100 1900 2045.65% (7 EOs) + 54.35% (60 EOs) (Composition V) Ethoxylated castor oil12 100 1900 20 34.78% (7 EOs) + 65.22% (60EOs) (Composition W)Ethoxylated castor oil 13 100 1900 20 23.91% (7 EOs) + 76.09% (60 EOs)(Composition X) Ethoxylated castor oil 14 100 1900 20 13.04% (7 EOs) +86.96% (60 EOs) (Composition Y) Mannitan oleate (15 EOs) 14.6 100 190020 (Composition J) Ethoxylated castor oil 15.2 100 1900 20 100% (60EOs)(Composition Z) Control (C1) 1000 1000 20 Control (C2) 0 2000 20

The results of the ELISA assays are as follows:

IgG1 assay (timescale in days) Composition D14 D28 D42 D56 D90 D180Composition (P) 1600 600 8000 16000 12000 nd Composition (Q) 3200 60016000 64000 48000 nd Composition (R) 2400 400 48000 64000 64000 ndComposition (S) nd nd nd nd nd nd Composition (T) 600 100 16000 3200032000 nd Composition (U) 100 100 8000 32000 32000 nd Composition (V) 100100 8000 12000 6000 nd Composition (W) nd nd nd nd nd nd Composition (X)200 100 3000 4000 3000 nd Composition (Y) 400 100 4000 12000 8000 ndComposition (Z) 100 100 8000 6000 6000 nd Control (C1) 19200 12800256000 128000 128000 nd Control (C2) 100 100 4000 2000 1500 nd

IgG2a assay (timescale in days) Composition D14 D28 D42 D56 D90 D180Composition (P) 100 100 8000 3000 3000 nd Composition (Q) 100 100 12004000 8000 nd Composition (R) 100 100 32000 8000 8000 nd Composition (S)nd nd nd nd nd nd Composition (T) 100 100 16000 3000 8000 nd Composition(U) 100 100 6000 4000 4000 nd Composition (V) 100 100 4000 15000 2000 ndComposition (W) nd nd nd nd nd nd Composition (X) 100 100 1000 1000 1000nd Composition (Y) 100 100 1000 1000 1000 nd Composition (Z) 100 1001000 1000 3000 nd Control C1 100 100 8000 3000 8000 nd Control C2 100100 1000 1000 1000 nd

1. A vaccine comprising: (i) at least one antigen or at least one invivo generator of a compound comprising an amino acid sequence, and (ii)a surfactant, or a mixture of surfactants, having an overall HLB numberof between 5 and 15 and comprising: ethoxylated derivatives of ester offatty acids having 12 to 22 carbon atoms with sorbitan or mannitanhaving a number of EOs of between 1 and 60; or ethoxylated derivativesof oils having a number of EOs of between 1 and
 60. 2. The vaccine asdefined in claim 1, wherein said vaccine does not include an oily phase.3. The vaccine as defined in claim 1, wherein said vaccine is suitablefor mucosal vaccination.
 4. The vaccine as defined in claim 1, whereinsaid vaccine suitable for application orally, nasally, rectally, orvaginally.
 5. The vaccine of claim 1, wherein said surfactant or saidmixture of surfactants comprises: ethoxylated derivatives of mannitanoleate having a number of EOs of between 5 and 15; ethoxylated derivatesof corn oil having a number of EOs between 20 and 40; or ethoxylatedderivatives of castor oil having a number of ECs equal to 7 or equal to60.